In the related art, as a cell culture substrate such as an animal tissue, a plastic (for example, polystyrene) container has been widely used. In order that the plastic container allows the cell culture to be effectively performed, a surface thereof is subjected to a plasma treatment or a surface treatment which is performed through the coating by using silicon or a cell attachment factor. In a case where such a cell culture container is used as the culture substrate, the cultured (proliferated) cells are attached on the surface of the container, and in order to detach and collect the cells, it is necessary to separate the cells from the surface of the container by using protein hydrolase such as trypsin or chemicals. An operation of separating the cells by using such enzyme and chemicals is a complicated step, and it is likely that impurities such as bacteria, DNA, and RNA are mixed. In addition, not only a bonding part between the cells and substrate is debonded, but also the bonding between the cells is debonded. For this reason, there is a problem in that the cells in a state of being proliferated (for example, a sheet state) cannot be extracted or are damaged, which causes the change of the cell properties.
In recent years, a technique in which the surface of the cell culture container is very thinly coated with a polymer having a lower critical solution temperature, such as poly-N-isopropyl acrylamide, the polymer exhibits the hydrophobicity at the cell culture temperature, the cells are attached to the polymer, after the cells are cultured, the adhesiveness between the cells and the polymer is decreased by subjecting the polymer to a low temperature treatment, and then the cells are separated in a sheet state from the substrate without using the hydrolytic enzyme and the chemicals was reported (for example, refer to PTL 1 and PTL 2).
However, a polymer such as poly-N-isopropyl acrylamide has low adhesiveness to a surface of plastic such as polystyrene, and when contacting with water, a coated polymer layer is easily detached. Thus, it is necessary to fix the polymer so as not to detach such a polymer layer from the surface of the plastic even in a case of contact with water. As one method for fixing the polymer, there is a method for allowing the surface of the cell culture substrate to be coated with a solution of N-isopropyl acrylamide (monomer) and then performing graft polymerization by irradiation of electron beams (for example, refer to PTL 3).
With respect to the graft polymerization by irradiation of electron beams, the cross-linking reaction between the polymers is necessarily caused on the polymerization, and with the progress of the degree of crosslinking, a temperature response rate of the polymer is greatly decreased, thereby causing a problem that a long period of time for maintaining a low temperature is required in order to make the polymer hydrophilic, and a problem that the cells are damaged during that time since they are also exposed at a low temperature for a long period of time. In addition, when the cell culture substrate produced by using the method is subjected to a sterilization treatment with radiation (for example, γ line), the temperature responsivity of the polymer becomes greatly decreased, which causes a problem that the original ability with which the cells are apt to detach therefrom is missed.
On the other hand, a technique in which a cell culture substrate containing a polymer (A) of a (meth)acrylic acid ester monomer (a), an inorganic material (C), and a polymer (B) having a lower critical solution temperature has excellent culturability with respect to various types of cells and properties for easily detaching the cultured cells by lowering the environmental temperature, and the culturability and the detachability can be easily controlled depending on the cell type was reported (for example, refer to PTL 4).
The polymer (B) having the lower critical solution temperature which is used in the above-described invention is a single polymer mainly containing poly-N-isopropyl acrylamide, and the lower critical solution temperature is also a single temperature, and thus it is not possible to freely change (control) the lower critical solution temperature.